Production of Rhizopus oryzae lipase using optimized Yarrowia lipolytica expression system

Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys(-)), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg). A novel expression platform was developed for erythritol-inducible expression of lipase from Rhizopus oryzae in Yarrowia lipolytica.

Automated summary

Yarrowia lipolytica is a suitable yeast for producing foreign proteins. A set of 18 chromogenic cloning vectors were developed, each containing an excisable auxotrophic selective marker and a different promoter. An improved recipient strain was developed by eliminating filamentation and introducing an auxotrophy for lysine. The optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined and higher lipase activity was achieved with certain promoters.